The LDSB investigates the organization and activities of developmental regulatory networks using formation of the Drosophila embryonic heart and body wall muscles as a model system. The overarching goal of this work is to comprehensively identify and characterize the upstream regulators of cell fate specification, the downstream effectors of differentiation, and the complex functional interactions that occur among these components during organogenesis. To achieve this objective, we combine contemporary genome-wide experimental and computational approaches with classical genetics and embryology to generate mechanistic hypotheses that we then test at single cell resolution in the intact organism. In several independent projects, we have applied different computational methods to suggest the involvement of particular transcription factors (TFs) in the regulation of gene expression in various subtypes of mesodermal cells. Moreover, we have used cis and trans experimental approaches to validate these hypotheses with particular transcriptional enhancers as test cases. To generalize these findings on the more global scale of cell type-specific gene regulatory networks, we have begun using chromatin immunoprecipitation (ChIP) to determine the in vivo localization of various TFs and modified histones in chromatin isolated from purified Drosophila embryonic mesodermal cells. Whereas other groups have undertaken ChIP experiments on whole embryos to identify targets regulated by such broadly expressed mesodermal TFs as Twist, Tinman and Mef2, this method has not been extensively used to study the role of TFs that confer individual muscle or heart cell identities since it is difficult to obtain adequate signals from the small number of cells in question. To address this problem at single cell resolution will require enriching for the cell types of interest, an approach that we have initiated using similar methods that we developed for genome-wide transcriptome profiling. To this end, we have modified standard ChIP-Seq protocols by starting with chromatin isolated from flow-sorted stage 11 mesodermal cells, and we are currently using informative antibodies to profile histone marks and bound TFs in such preparations. Information from these studies will then be incorporated into other projects to identify and validate cell type-specific enhancers, and to iteratively refine computational approaches for enhancer prediction. For example, we used the above approach in a separately described project in which we investigated the role of binding sequences that are preferentially bound by different members of the homedomain (HD) family in determining the regulatory specificity of the muscle founder cell HD TF Slouch (Slou). In this case, we asked whether Slou directly regulates a particular FC enhancer that we independently demonstrated contains a functional Slou-preferred binding site by using ChIP followed by quantitative real-time polymerase chain reaction (ChIP-qPCR) to definitively show that a genomic sequence that includes the Slou-preferred binding site in the enhancer in question is indeed bound by Slou in purified primary mesodermal cells. This result established that Slou binds to the relevant FC enhancer in vivo, and is consistent with the possibility that Slou directly regulates this element through Slou-preferred binding sites.